短发夹状RNA基因沉默补体受体C5aR及抑制LPS诱导的肾脏上皮细胞凋亡

Small hairpin RNA targeting rat C5aR can inhibit the cell apoptosis induced by LPS in kidney epithelial cells

目的:探讨短发夹状RNA基因沉默补体受体C5aR及抑制LPS诱导的肾脏上皮细胞凋亡的作用效果。方法:构建针对大鼠补体受体C5aR基因编码区的短发夹状RNA(shRNA)真核表达载体质粒pRNAT-U6.1-C5aR shRNA,采用电穿孔的方法转染RK3E细胞,经G418筛选后,形成稳定的表达C5aR shRNA的细胞系。实验分为3组,①正常对照组:未转染的RK3E细胞;②阴性对照组:转染空载体pRNAT-U6.1的RK3E细胞系;③实验组:转染C5aR shRNA的RK3E细胞系。经脂多糖(LPS)孵育12小时后,流式细胞仪检测各组细胞凋亡率,RT-PCR检测mRNA的表达,γ计数仪测定125I标记的C5a与RK3E的结合情况。结果:与正常对照组和阴性对照组相比,实验组的细胞凋亡率显著降低(P<0.01),C5aR mRNA水平显著降低(P<0.01),125I标记的C5a与RK3E结合活性显著下降。结论:针对C5aR的特异性短发夹RNA可以明显引起靶基因的沉默,进而抑制LPS诱导的肾脏上皮细胞凋亡的发生。

Objective：To investigate RNA interference and apoptosis induced by LPS in kidney epithelial cells through silencing CSaR gene with small hairpin RNA （shRNA）. Methods：We construct the eukaryotic expression vector of small hairpin RNA targeting rat C5aR gene, and transfected RK3E cell by electroporation,after G418 selection,so we got the stable cell line expressing CSaR shRNA.The experiment was designed into 3 groups：Onormal control group, RK3E cells without transfection; Qnegative control group, RK3E cells transfected with blank vector; （3） experimental group, RK3E cells transfected with CSaR shRNA. After incubation with LPS for 12 h, the ratio of apoptosis was tested by flow cytometry, the level of mRNA was tested by RT-PCR, and binding of 125I-rrC5a to RK3E cells stimulated with LPS were performed to examine the expression of CSaR in RK3E cells. Results： Compared with the normal control group and negative control group, in the experimental group the ratio of apoptosis was significantly decreased（ P 〈 0.01 ）, and the expression of CSaR mRNA was significantly inhibited（ P 〈 0.01 ）, and binding of 125I-nC5a to RK3E cells was significantly decreased also. Conclusion： Hairpin shRNA targeting CSaR gene can lead to obvious gene silence in vitro and inhibit the cell apoptosis induced by LPS in kidney epithelial cell.